The proposed work centers on two areas of research of the adrenocortical steroid 11 beta-hydroxylase as a model system for studying cytochrome P-450 catalyzed mixed function oxidations (such as aerobic hydroxylations and demethylations): 1) Preparation and purification of the three components of the adrenocortical steroid 11 beta-hydroxylase: flavoprotein(Fp), iron sulfur protein(ISP), and the heme protein P-450. 2) Chemical and physical characterization of these components - These investigations will include: optical and EPR spectral studies; rapid reaction kinetics of interaction of stoichiometrically reduced components with the appropriate oxidized or oxygenated form; chemical composition of these components; study of mechanism of interaction of steroid substrates, sterol-related steroids and hydroxylase inhibitors with heme P-450; tests for the requirements for various entities such as sterols, phospholipids, steroid-sterol carrier proteins, etc., for restoration of full hydroxylase activity of the reconstituted system; study of the mechanism for the indispensable requirement for reduced iron sulfur protein. The overall goal is to obtain an understanding of the mechanism of oxygen activation in aerobic hydroxylations catalyzed by P-450 dependent mixed function oxidase systems which are widely distributed throughout the body and are catalyzing important steps required for metabolism of steroids, drugs, xenobiotics, carcinogens, and the biosynthesis of androgens, estrogens and adrenocortical steroids.